Studies of the virulence of Bordetella avium revealed a novel cytotoxic protein, designated osteotoxin, that is lethal for MC3T3-E1 osteogenic cells, fetal bovine trabecular cells, embryonic bovine tracheal cells, and UMR106-01(BSP) rat osteo- sarcoma cells. The metabolic activity of IC-21 and ANA-1 mouse macrophage, and J774A.1 monocyte-macrophage cell lines is partially inhibited by the osteotoxin, whereas RAW264.7 mouse monocyte-macrophage cell cultures and L6 myoblasts are relatively unaffected. The osteotoxin lacks both hemolytic and dermonecrotic toxin activities, and is non-proteolytic. Osteotoxin was purified to homogeneity from B. avium 197 by conventional chromatographic procedures. The native protein (Mr 80,000; pI 4.5) is a homodimer, and the two, non-covalently linked subunits contain a pyridoxal phosphate co-factor. The N-terminal amino acid sequence of the osteotoxin was obtained and two oligonucleotides were generated for use as probes in screening a B. avium gene library. A B. avium gene library was constructed in E. coli and recombinant clones were identified that contained a recombinant plasmid, p4-25, which encoded B. avium osteotoxin. DNA sequence analysis of the osteotoxin gene indicated that it is composed of 1191 basepairs of DNA and encodes a protein with a molecular mass of 42,600. The B. avium osteotoxin is highly homologous (38% identity and 60% similarity) to cystathioninase of E. coli and Salmonella typhimurium. Exposure of MC3T3-E1 osteogenic cells to B. avium osteotoxin (cystathionase) in the presence of 35S-cystine resulted in specific labeling of one major cell protein. We propose that B. avium cystathionase causes cell death by hydrolysis of cystine and generation of reactive sulfane sulfurs which inactivate or overactivate essential cellular enzymes. Seven Fusobacterium species were examined for the presence of a protein(s) which reacts with antibody against B. avium cystathionase. In this test, immunologically related proteins were found in, F. periodonticum, F. necrophorum, F. necrogenes, F. varium and F. mortiferum, but not in F. nucleatum and F. russii. Cystathionase purified from F. mortiferum was lethal for MC3T3-E1 osteogenic cells.